Antisense modulation of Her-3 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for inhibiting the expression of Her-3. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Her-3. Methods of using these compounds for inhibition of Her-3 expression and for treatment of diseases associated with expression of Her-3 are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of Her-3. In particular, this invention relates tocompounds, particularly oligonucleotides, specifically hybridizable withnucleic acids encoding Her-3. Such compounds have been shown to modulatethe expression of Her-3.

BACKGROUND OF THE INVENTION

One of the principal mechanisms by which cellular regulation is effectedis through the transduction of extracellular signals into intracellularsignals that in turn modulate biochemical pathways. Examples of suchextracellular signaling molecules include growth factors, cytokines, andchemokines. The cell surface receptors of these molecules and theirassociated signal transduction pathways are therefore one of theprincipal means by which cellular behavior is regulated. Becausecellular phenotypes are largely influenced by the activity of thesepathways, it is currently believed that a number of disease statesand/or disorders are a result of either aberrant activation orfunctional mutations in the molecular components of signal transductionpathways. Consequently, considerable attention has been devoted to thecharacterization of these receptor proteins.

HER3 (also known as ErbB3), a member of the EGF family ofreceptor/tyrosine kinases, is a protein that has been shown to play acomplex role in several signal transduction pathways by forming homo-and heterodimers with other members of the EGF family depending on theirconcentrations and the concentration of particular ligands. Unlike othermembers of the family, HER3 lacks an intrinsic kinase activity and mustrely on the presence of HER2 to transduce the signal across themembrane. However, HER3 can bind ATP and can recruit SH2-containingproteins (Carraway, BioEssays, 1996, 18, 263-266). The two groups ofligands specific to HER3 and HER4 are collectively termed neuregulins(NRGs) because of their demonstrated role in the nervous system. HER3and HER4 function as the binding receptors; and HER2 along with EGFR areconsidered co-receptors and are recruited as partners to HER3 and HER4upon ligand binding. (Burden and Yarden, Neuron, 1997, 18, 847-855).

HER3, first cloned in 1989, was found in a variety of normal epithelialtissues as well as being overexpressed in human mammary tumor cell lines(Kraus et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 9193-9197). It hasalso been shown to act in a variety of cellular signaling cascadesincluding those involved in neural development (Britsch et al., GenesDev., 1998, 12, 1825-1836), mammary morphogenesis (Alimandi et al.,Oncogene, 1995, 10, 1813-1821), and phosphinositide 3-kinase p85 binding(Hellyer et al., Biochem. J., 1998, 333, 757-763). Disclosed in U.S.Pat. No. 5,183,884 and in the European Patent Application 0 444 961 A1are the DNA encoding HER-3 as well as probes for HER-3, vectors encodingHER-3 and cell cultures expressing such vectors (Kraus and Aaronson,1993; Plowman et al., 1991).

Manifestations of altered HER3 regulation appear in both injury anddisease states, the most important being in the development of cancer.Cellular transformation and acquisition of the metastatic phenotype arethe two main changes normal cells undergo during the progression tocancer. HER3 in cooperation with HER2 has been shown to be involved inthe neoplastic transformation of cells (Alimandi et al., Oncogene, 1995,10, 1813-1821). Recently, it has been demonstrated that HER-3 and HER-4are expressed at high levels in gastric cancers with three out of sixgastric cancers expression HER-3 and four out of six gastric cancersoverexpressing HER-4 (Kataoka et al., Life Sci., 1998, 63, 553-564).

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of HER3. Consequently, there remains a long feltneed for additional agents capable of effectively inhibiting HER3function.

To date, strategies aimed at inhibiting HER3 function have involved theuse of antibodies, antisense to the HER3 coreceptor, HER2, geneknockouts of HER2 and HER3 in mice and modifications to the ligands thatbind HER3.

Studies using antisense to reduce the expression of HER2, the coreceptorof HER3 showed that reduced HER2 attenuated the effect of HER3 signalingbut did not abolish it (Yoo and Hamburger, Mol. Cell. Endocrinol., 1998,138, 163-171). Mice lacking HER3 die in utero due to the thinned andrudimentary development of the atrioventricular valves of the heart(Burden and Yarden, Neuron, 1997, 18, 847-855).

Therefore, it appears as though these targeting strategies are eitherlethal or lack specificity, thus warranting further development ofentities capable of safely inhibiting HER3 function. Antisenseoligonucleotides provide a promising new pharmaceutical tool for theeffective modification of the expression of specific genes.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, particularly antisenseoligonucleotides, which are targeted to a nucleic acid encoding Her-3,and which modulate the expression of Her-3. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided. Further provided are methods of modulating the expression ofHer-3 in cells or tissues comprising contacting said cells or tissueswith one or more of the antisense compounds or compositions of theinvention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of Her-3 by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding Her-3, ultimately modulating the amountof Her-3 produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingHer-3. As used herein, the terms “target nucleic acid” and “nucleic acidencoding Her-3” encompass DNA encoding Her-3, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. The specific hybridization of an oligomeric compound with itstarget nucleic acid interferes with the normal function of the nucleicacid. This modulation of function of a target nucleic acid by compoundswhich specifically hybridize to it is generally referred to as“antisense”. The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of Her-3. In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding Her-3. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding Her-3, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense and other compounds of the invention which hybridize to thetarget and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 50 nucleobases (i.e.from about 8 to about 50 linked nucleosides). Particularly preferredantisense compounds are antisense oligonucleotides, even more preferablythose comprising from about 12 to about 30 nucleobases. Antisensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—,S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, also known as2′—O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference, and U.S. Pat. No. 5,750,692, which is commonly owned withthe instant application and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of Her-3 is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingHer-3, enabling sandwich and other assays to easily be constructed toexploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding Her-3 can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of Her-3 in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/ cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1), or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and2′-alkoxy amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham MA or GlenResearch, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

21′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites 2′-O-Methoxyethyl-substitutednucleoside amidites are prepared as follows, or alternatively, as perthe methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue as dissolved inMeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×L) andbrine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was stirred for 1 h. Solvent was removed undervacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.Nos. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxy ethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing 5 cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, Ribonucleaseprotection assays, or RT-PCR.

T-24 Cells

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

MCF-7 Cells

The human breast carcinoma cell line MCF-7 was obtained from theAmerican Type Culure Collection (Manassas, Va.). MCF-7 cells wereroutinely cultured in DMEM low glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-ME™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf MRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

Analysis of Oligonucleotide Inhibition of Her-3 Expression

Antisense modulation of Her-3 expression can be assayed in a variety ofways known in the art. For example, Her-3 mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+MRNA. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calf. and used according to manufacturer'sinstructions. Prior to quantitative PCR analysis, primer-probe setsspecific to the target gene being measured are evaluated for theirability to be “multiplexed” with a GAPDH amplification reaction. Inmultiplexing, both the target gene and the internal standard gene GAPDHare amplified concurrently in a single sample. In this analysis, MRNAisolated from untreated cells is serially diluted. Each dilution isamplified in the presence of primer-probe sets specific for GAPDH only,target gene only (“single-plexing”), or both (multiplexing). FollowingPCR amplification, standard curves of GAPDH and target mRNA signal as afunction of dilution are generated from both the single-plexed andmultiplexed samples. If both the slope and correlation coefficient ofthe GAPDH and target signals generated from the multiplexed samples fallwithin 10% of their corresponding values generated from thesingle-plexed samples, the primer-probe set specific for that target isdeemed as multiplexable. Other methods of PCR are also known in the art.

Protein levels of Her-3 can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to Her-3 can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11

Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

Real-time Quantitative PCR Analysis of Her-3 mRNA Levels

Quantitation of Her-3 MRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE, FAM, or VIC, obtained from either Operon TechnologiesInc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) MRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

Probes and primers to human Her-3 were designed to hybridize to a humanHer-3 sequence, using published sequence information (GenBank accessionnumber M34309, incorporated herein as SEQ ID NO:3). For human Her-3 thePCR primers were:

forward primer: TCGTCATGTTGAACTATAACACCAACT (SEQ ID NO: 4) reverseprimer: TGACAAAGCTTATCGTTCTTCTCAAT (SEQ ID NO: 5) and the PCR probe was:FAM-CCGCTTGACTCAGCTCACCGAGATTC-TAMRA (SEQ ID NO: 6) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For human GAPDH the PCR primers were:

forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7) reverse primer:GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 8) and the PCR probe was: 5′JOE-CGCCTGGTCACCAGGGCTGCT-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

Northern Blot Analysis of Her-3 mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human Her-3, a human Her-3 specific probe was prepared by PCRusing the forward primer TCGTCATGTTGAACTATAACACCAACT (SEQ ID NO: 4) andthe reverse primer TGACAAAGCTTATCGTTCTTCTCAAT (SEQ ID NO: 5). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

Antisense Inhibition of Human Her-3 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE wings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human Her-3 RNA, usingpublished sequences (GenBank accession number M34309, incorporatedherein as SEQ ID NO: 3, and GenBank accession number U75285,incorporated herein as SEQ ID NO: 10). The oligonucleotides are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 18nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by four-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human Her-3 mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human Her-3 mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO19547 5′ UTR 3  71 cgacggcagcggaggttg 28 11 19548 5′ UTR 3  112aagagccacctgaaccgc 57 12 19549 5′ UTR 3  176 cgcagagggtgaagggag  4 1319550 Coding 3  253 gttgcccacctcggagcc 59 14 19551 Coding 3  266acactgcctgagagttgc 43 15 19552 Coding 3  326 gtgtctggtattggttct 61 1619553 Coding 3  360 atcaccacctcacacctc 65 17 19554 Coding 3  390tgtcccgtgagcacaatc 41 18 19555 Coding 3  403 gaggtcggcattgtgtcc 24 1919556 Coding 3  414 tgcaggaaggagaggtcg 48 20 19557 Coding 3  490gcggaggttgggcaatgg 37 21 19558 Coding 3  569 cgtggctggagttggtgt 96 2219559 Coding 3  615 cctgacagaatctcggtg 87 23 19560 Coding 3  689ggtccctcacgatgtccc 80 24 19561 Coding 3  706 cactatctcagcatctcg 59 2519562 Coding 3  720 ccattgtccttcaccact 36 26 19563 Coding 3  772aggaccccagcatcgccc 65 27 19564 Coding 3  797 tcaatgtctggcagtctt 63 2819565 Coding 3  813 gcacagatggtcttggtc 80 29 19566 Coding 3  828ccattacactgaggagca 57 30 19567 Coding 3  866 catggcagcactggttgg 65 3119568 Coding 3  918 caggcaaagcagtctgtg 48 32 19569 Coding 3  977tgtagacaagaggctgtg 49 33 19570 Coding 3 1039 acaaactcctccatactg 33 3419571 Coding 3 1089 ctgacacaggatgtttga 37 35 19572 Coding 3 1139acatcttgagcccatttt 44 36 19573 Coding 3 1191 ccagagcctgttccctca 38 3719574 Coding 3 1239 ttcacaaatccatcaatg 10 38 19575 Coding 3 1383gactggatgttcaggtaa 72 39 19576 Coding 3 1458 cggttgtagaggcttctg 24 4019577 Coding 3 1513 tcggaagcccagagatgt 53 41 19578 Coding 3 1582agagtggtggtagcagag  5 42 19579 Coding 3 1603 aagcaccttggtccagtt 67 4319580 Coding 3 1738 ggacaagcactgaccagg 49 44 19581 Coding 3 1839tggcaggagaagcattcg 32 45 19582 Coding 3 1900 acaagtatcagagcccga 60 4619583 Coding 3 1913 gggcacattgagcacaag 11 47 19584 Coding 3 2076tgtcctaaacagtcttga 41 48 19585 Coding 3 2110 tgtcagatgggttttgcc 46 4919586 Coding 3 2133 cctgctatcactgtcaaa  7 50 19587 Coding 3 2146aatcactaccaatcctgc  0 51 19588 Coding 3 2179 ccagtagagaaaagtgcc 46 5219589 Coding 3 2215 cctcatagcccttttatt 51 53 19590 Coding 3 2232ccccgttccaagtatcgc 47 54 19591 Coding 3 2242 tatgctctcaccccgttc 14 5519592 Coding 3 2278 gactttgttagccttctc 68 56 19593 Coding 3 2339agacacccgagccaagca 30 57 19594 Coding 3 2388 ttgattgattcaccctca 72 5819595 Coding 3 2427 ccactcttgtcctcaatg 72 59 19596 Coding 3 2440aaaactctgccgtccact 43 60 19597 Coding 3 2512 tagtcccagcagccttac 42 6119598 Coding 3 2564 gagaacccagaggcaaat 54 62 19599 Coding 3 2627ctccccagttgagcagca 70 63 19600 Coding 3 2694 cgggcagccaggtttcta 65 6419601 Coding 3 2731 cacctgaacctgactggg 43 65 19602 Coding 3 2744caccaaaatctgccacct 59 66 19603 Coding 3 2770 atcatcaggaggcagcag 52 6719604 Coding 3 2839 cccaaagtggatactctc 73 68 19605 Coding 3 2893caactcccaaactgtcac 69 69 19606 Coding 3 2906 ccccgaaggtcatcaact 36 7019607 Coding 3 2943 ggtacttcagccaatcgt 62 71 19608 Coding 3 3034atcaatcatccaacactt 12 72 19609 Coding 3 3048 gggcgaatgttctcatca 30 7319610 Coding 3 3178 cttcttgtttgtcagacc 77 74 19611 Coding 3 3293gtgttccaactggtaggc 20 75 19612 Coding 3 3446 gtggcattgggtgtagag 43 7619613 Coding 3 3524 acattgacactttctcct 46 77 19614 Coding 3 3654ccgttgacatcctcttcc 30 78 19615 Coding 3 3736 gacagaactgagacccac 35 7919616 Coding 3 3776 attcatactcctcatctt 21 80 19617 Coding 3 3957tcttcatctggagttgtg 55 81 19618 Coding 3 4051 ttcatacccttgctcaga 56 8219619 Coding 3 4161 tcagggttatcaaaggca 64 83 19620 Coding 3 4209tacgttctctgggcatta 54 84 19621 3′ UTR 3 4353 agcctccaaagattgaat 35 8519622 3′ UTR 3 4467 ggaatgggactgggaaag 16 86 19623 3′ UTR 3 4572acaaaggcacacataaga 34 87 19624 3′ UTR 3 4603 ccctttcctcttcttgac 29 8819625 3′ UTR 3 4697 tcctaacctctttcttca  0 89 19626 3′ UTR 3 4766gtaggtgaagtttagtat 53 90 19627 3′ UTR 3 4794 aggatgataaaggactaa  2 9119629 3′ UTR 3 4928 tggtctcaaactccttgc 39 92 19630 3′ UTR 3 4951gggtcttactatgttggc 60 93 19628 Intron 10  6560 caaagtgctgagattaca 29 94

As shown in Table 1, SEQ ID NOs 11, 12, 14, 15, 16, 17, 18, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 41, 43,44, 45, 46, 48, 49, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 87,88, 90, 92, 93 and 94 demonstrated at least 25% inhibition of humanHer-3 expression in this assay and are therefore preferred. The targetsites to which these preferred sequences are complementary are hereinreferred to as “active sites” and are therefore preferred sites fortargeting by compounds of the present invention.

Example 16

Western Blot Analysis of Her-3 Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to Her-3 is used, with aradiolabelled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 94 <210> SEQ ID NO 1 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2atgcattctg cccccaagga 20 <210> SEQ ID NO 3 <211> LENGTH: 4975 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (199)...(4227) <400> SEQUENCE: 3 ctctcacacacacacacccc tcccctgcca tccctccccg gactccggct ccggctccga 60 ttgcaatttgcaacctccgc tgccgtcgcc gcagcagcca ccaattcgcc agcggttcag 120 gtggctcttgcctcgatgtc ctagcctagg ggcccccggg ccggacttgg ctgggctccc 180 ttcaccctctgcggagtc atg agg gcg aac gac gct ctg cag gtg ctg ggc 231 Met Arg Ala AsnAsp Ala Leu Gln Val Leu Gly 1 5 10 ttg ctt ttc agc ctg gcc cgg ggc tccgag gtg ggc aac tct cag gca 279 Leu Leu Phe Ser Leu Ala Arg Gly Ser GluVal Gly Asn Ser Gln Ala 15 20 25 gtg tgt cct ggg act ctg aat ggc ctg agtgtg acc ggc gat gct gag 327 Val Cys Pro Gly Thr Leu Asn Gly Leu Ser ValThr Gly Asp Ala Glu 30 35 40 aac caa tac cag aca ctg tac aag ctc tac gagagg tgt gag gtg gtg 375 Asn Gln Tyr Gln Thr Leu Tyr Lys Leu Tyr Glu ArgCys Glu Val Val 45 50 55 atg ggg aac ctt gag att gtg ctc acg gga cac aatgcc gac ctc tcc 423 Met Gly Asn Leu Glu Ile Val Leu Thr Gly His Asn AlaAsp Leu Ser 60 65 70 75 ttc ctg cag tgg att cga gaa gtg aca ggc tat gtcctc gtg gcc atg 471 Phe Leu Gln Trp Ile Arg Glu Val Thr Gly Tyr Val LeuVal Ala Met 80 85 90 aat gaa ttc tct act cta cca ttg ccc aac ctc cgc gtggtg cga ggg 519 Asn Glu Phe Ser Thr Leu Pro Leu Pro Asn Leu Arg Val ValArg Gly 95 100 105 acc cag gtc tac gat ggg aag ttt gcc atc ttc gtc atgttg aac tat 567 Thr Gln Val Tyr Asp Gly Lys Phe Ala Ile Phe Val Met LeuAsn Tyr 110 115 120 aac acc aac tcc agc cac gct ctg cgc cag ctc cgc ttgact cag ctc 615 Asn Thr Asn Ser Ser His Ala Leu Arg Gln Leu Arg Leu ThrGln Leu 125 130 135 acc gag att ctg tca ggg ggt gtt tat att gag aag aacgat aag ctt 663 Thr Glu Ile Leu Ser Gly Gly Val Tyr Ile Glu Lys Asn AspLys Leu 140 145 150 155 tgt cac atg gac aca att gac tgg agg gac atc gtgagg gac cga gat 711 Cys His Met Asp Thr Ile Asp Trp Arg Asp Ile Val ArgAsp Arg Asp 160 165 170 gct gag ata gtg gtg aag gac aat ggc aga agc tgtccc ccc tgt cat 759 Ala Glu Ile Val Val Lys Asp Asn Gly Arg Ser Cys ProPro Cys His 175 180 185 gag gtt tgc aag ggg cga tgc tgg ggt cct gga tcagaa gac tgc cag 807 Glu Val Cys Lys Gly Arg Cys Trp Gly Pro Gly Ser GluAsp Cys Gln 190 195 200 aca ttg acc aag acc atc tgt gct cct cag tgt aatggt cac tgc ttt 855 Thr Leu Thr Lys Thr Ile Cys Ala Pro Gln Cys Asn GlyHis Cys Phe 205 210 215 ggg ccc aac ccc aac cag tgc tgc cat gat gag tgtgcc ggg ggc tgc 903 Gly Pro Asn Pro Asn Gln Cys Cys His Asp Glu Cys AlaGly Gly Cys 220 225 230 235 tca ggc cct cag gac aca gac tgc ttt gcc tgccgg cac ttc aat gac 951 Ser Gly Pro Gln Asp Thr Asp Cys Phe Ala Cys ArgHis Phe Asn Asp 240 245 250 agt gga gcc tgt gta cct cgc tgt cca cag cctctt gtc tac aac aag 999 Ser Gly Ala Cys Val Pro Arg Cys Pro Gln Pro LeuVal Tyr Asn Lys 255 260 265 cta act ttc cag ctg gaa ccc aat ccc cac accaag tat cag tat gga 1047 Leu Thr Phe Gln Leu Glu Pro Asn Pro His Thr LysTyr Gln Tyr Gly 270 275 280 gga gtt tgt gta gcc agc tgt ccc cat aac tttgtg gtg gat caa aca 1095 Gly Val Cys Val Ala Ser Cys Pro His Asn Phe ValVal Asp Gln Thr 285 290 295 tcc tgt gtc agg gcc tgt cct cct gac aag atggaa gta gat aaa aat 1143 Ser Cys Val Arg Ala Cys Pro Pro Asp Lys Met GluVal Asp Lys Asn 300 305 310 315 ggg ctc aag atg tgt gag cct tgt ggg ggacta tgt ccc aaa gcc tgt 1191 Gly Leu Lys Met Cys Glu Pro Cys Gly Gly LeuCys Pro Lys Ala Cys 320 325 330 gag gga aca ggc tct ggg agc cgc ttc cagact gtg gac tcg agc aac 1239 Glu Gly Thr Gly Ser Gly Ser Arg Phe Gln ThrVal Asp Ser Ser Asn 335 340 345 att gat gga ttt gtg aac tgc acc aag atcctg ggc aac ctg gac ttt 1287 Ile Asp Gly Phe Val Asn Cys Thr Lys Ile LeuGly Asn Leu Asp Phe 350 355 360 ctg atc acc ggc ctc aat gga gac ccc tggcac aag atc cct gcc ctg 1335 Leu Ile Thr Gly Leu Asn Gly Asp Pro Trp HisLys Ile Pro Ala Leu 365 370 375 gac cca gag aag ctc aat gtc ttc cgg acagta cgg gag atc aca ggt 1383 Asp Pro Glu Lys Leu Asn Val Phe Arg Thr ValArg Glu Ile Thr Gly 380 385 390 395 tac ctg aac atc cag tcc tgg ccg ccccac atg cac aac ttc agt gtt 1431 Tyr Leu Asn Ile Gln Ser Trp Pro Pro HisMet His Asn Phe Ser Val 400 405 410 ttt tcc aat ttg aca acc att gga ggcaga agc ctc tac aac cgg ggc 1479 Phe Ser Asn Leu Thr Thr Ile Gly Gly ArgSer Leu Tyr Asn Arg Gly 415 420 425 ttc tca ttg ttg atc atg aag aac ttgaat gtc aca tct ctg ggc ttc 1527 Phe Ser Leu Leu Ile Met Lys Asn Leu AsnVal Thr Ser Leu Gly Phe 430 435 440 cga tcc ctg aag gaa att agt gct gggcgt atc tat ata agt gcc aat 1575 Arg Ser Leu Lys Glu Ile Ser Ala Gly ArgIle Tyr Ile Ser Ala Asn 445 450 455 agg cag ctc tgc tac cac cac tct ttgaac tgg acc aag gtg ctt cgg 1623 Arg Gln Leu Cys Tyr His His Ser Leu AsnTrp Thr Lys Val Leu Arg 460 465 470 475 ggg cct acg gaa gag cga cta gacatc aag cat aat cgg ccg cgc aga 1671 Gly Pro Thr Glu Glu Arg Leu Asp IleLys His Asn Arg Pro Arg Arg 480 485 490 gac tgc gtg gca gag ggc aaa gtgtgt gac cca ctg tgc tcc tct ggg 1719 Asp Cys Val Ala Glu Gly Lys Val CysAsp Pro Leu Cys Ser Ser Gly 495 500 505 gga tgc tgg ggc cca ggc cct ggtcag tgc ttg tcc tgt cga aat tat 1767 Gly Cys Trp Gly Pro Gly Pro Gly GlnCys Leu Ser Cys Arg Asn Tyr 510 515 520 agc cga gga ggt gtc tgt gtg acccac tgc aac ttt ctg aat ggg gag 1815 Ser Arg Gly Gly Val Cys Val Thr HisCys Asn Phe Leu Asn Gly Glu 525 530 535 cct cga gaa ttt gcc cat gag gccgaa tgc ttc tcc tgc cac ccg gaa 1863 Pro Arg Glu Phe Ala His Glu Ala GluCys Phe Ser Cys His Pro Glu 540 545 550 555 tgc caa ccc atg ggg ggc actgcc aca tgc aat ggc tcg ggc tct gat 1911 Cys Gln Pro Met Gly Gly Thr AlaThr Cys Asn Gly Ser Gly Ser Asp 560 565 570 act tgt gct caa tgt gcc catttt cga gat ggg ccc cac tgt gtg agc 1959 Thr Cys Ala Gln Cys Ala His PheArg Asp Gly Pro His Cys Val Ser 575 580 585 agc tgc ccc cat gga gtc ctaggt gcc aag ggc cca atc tac aag tac 2007 Ser Cys Pro His Gly Val Leu GlyAla Lys Gly Pro Ile Tyr Lys Tyr 590 595 600 cca gat gtt cag aat gaa tgtcgg ccc tgc cat gag aac tgc acc cag 2055 Pro Asp Val Gln Asn Glu Cys ArgPro Cys His Glu Asn Cys Thr Gln 605 610 615 ggg tgt aaa gga cca gag cttcaa gac tgt tta gga caa aca ctg gtg 2103 Gly Cys Lys Gly Pro Glu Leu GlnAsp Cys Leu Gly Gln Thr Leu Val 620 625 630 635 ctg atc ggc aaa acc catctg aca atg gct ttg aca gtg ata gca gga 2151 Leu Ile Gly Lys Thr His LeuThr Met Ala Leu Thr Val Ile Ala Gly 640 645 650 ttg gta gtg att ttc atgatg ctg ggc ggc act ttt ctc tac tgg cgt 2199 Leu Val Val Ile Phe Met MetLeu Gly Gly Thr Phe Leu Tyr Trp Arg 655 660 665 ggg cgc cgg att cag aataaa agg gct atg agg cga tac ttg gaa cgg 2247 Gly Arg Arg Ile Gln Asn LysArg Ala Met Arg Arg Tyr Leu Glu Arg 670 675 680 ggt gag agc ata gag cctctg gac ccc agt gag aag gct aac aaa gtc 2295 Gly Glu Ser Ile Glu Pro LeuAsp Pro Ser Glu Lys Ala Asn Lys Val 685 690 695 ttg gcc aga atc ttc aaagag aca gag cta agg aag ctt aaa gtg ctt 2343 Leu Ala Arg Ile Phe Lys GluThr Glu Leu Arg Lys Leu Lys Val Leu 700 705 710 715 ggc tcg ggt gtc tttgga act gtg cac aaa gga gtg tgg atc cct gag 2391 Gly Ser Gly Val Phe GlyThr Val His Lys Gly Val Trp Ile Pro Glu 720 725 730 ggt gaa tca atc aagatt cca gtc tgc att aaa gtc att gag gac aag 2439 Gly Glu Ser Ile Lys IlePro Val Cys Ile Lys Val Ile Glu Asp Lys 735 740 745 agt gga cgg cag agtttt caa gct gtg aca gat cat atg ctg gcc att 2487 Ser Gly Arg Gln Ser PheGln Ala Val Thr Asp His Met Leu Ala Ile 750 755 760 ggc agc ctg gac catgcc cac att gta agg ctg ctg gga cta tgc cca 2535 Gly Ser Leu Asp His AlaHis Ile Val Arg Leu Leu Gly Leu Cys Pro 765 770 775 ggg tca tct ctg cagctt gtc act caa tat ttg cct ctg ggt tct ctg 2583 Gly Ser Ser Leu Gln LeuVal Thr Gln Tyr Leu Pro Leu Gly Ser Leu 780 785 790 795 ctg gat cat gtgaga caa cac cgg ggg gca ctg ggg cca cag ctg ctg 2631 Leu Asp His Val ArgGln His Arg Gly Ala Leu Gly Pro Gln Leu Leu 800 805 810 ctc aac tgg ggagta caa att gcc aag gga atg tac tac ctt gag gaa 2679 Leu Asn Trp Gly ValGln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Glu 815 820 825 cat ggt atg gtgcat aga aac ctg gct gcc cga aac gtg cta ctc aag 2727 His Gly Met Val HisArg Asn Leu Ala Ala Arg Asn Val Leu Leu Lys 830 835 840 tca ccc agt caggtt cag gtg gca gat ttt ggt gtg gct gac ctg ctg 2775 Ser Pro Ser Gln ValGln Val Ala Asp Phe Gly Val Ala Asp Leu Leu 845 850 855 cct cct gat gataag cag ctg cta tac agt gag gcc aag act cca att 2823 Pro Pro Asp Asp LysGln Leu Leu Tyr Ser Glu Ala Lys Thr Pro Ile 860 865 870 875 aag tgg atggcc ctt gag agt atc cac ttt ggg aaa tac aca cac cag 2871 Lys Trp Met AlaLeu Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln 880 885 890 agt gat gtctgg agc tat ggt gtg aca gtt tgg gag ttg atg acc ttc 2919 Ser Asp Val TrpSer Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe 895 900 905 ggg gca gagccc tat gca ggg cta cga ttg gct gaa gta cca gac ctg 2967 Gly Ala Glu ProTyr Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu 910 915 920 cta gag aagggg gag cgg ttg gca cag ccc cag atc tgc aca att gat 3015 Leu Glu Lys GlyGlu Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp 925 930 935 gtc tac atggtg atg gtc aag tgt tgg atg att gat gag aac att cgc 3063 Val Tyr Met ValMet Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg 940 945 950 955 cca accttt aaa gaa cta gcc aat gag ttc acc agg atg gcc cga gac 3111 Pro Thr PheLys Glu Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp 960 965 970 cca ccacgg tat ctg gtc ata aag aga gag agt ggg cct gga ata gcc 3159 Pro Pro ArgTyr Leu Val Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala 975 980 985 cct gggcca gag ccc cat ggt ctg aca aac aag aag cta gag gaa gta 3207 Pro Gly ProGlu Pro His Gly Leu Thr Asn Lys Lys Leu Glu Glu Val 990 995 1000 gag ctggag cca gaa cta gac cta gac cta gac ttg gaa gca gag gag 3255 Glu Leu GluPro Glu Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu 1005 1010 1015 gacaac ctg gca acc acc aca ctg ggc tcc gcc ctc agc cta cca gtt 3303 Asp AsnLeu Ala Thr Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Val 1020 1025 10301035 gga aca ctt aat cgg cca cgt ggg agc cag agc ctt tta agt cca tca3351 Gly Thr Leu Asn Arg Pro Arg Gly Ser Gln Ser Leu Leu Ser Pro Ser1040 1045 1050 tct gga tac atg ccc atg aac cag ggt aat ctt ggg ggg tcttgc cag 3399 Ser Gly Tyr Met Pro Met Asn Gln Gly Asn Leu Gly Gly Ser CysGln 1055 1060 1065 gag tct gca gtt tct ggg agc agt gaa cgg tgc ccc cgtcca gtc tct 3447 Glu Ser Ala Val Ser Gly Ser Ser Glu Arg Cys Pro Arg ProVal Ser 1070 1075 1080 cta cac cca atg cca cgg gga tgc ctg gca tca gagtca tca gag ggg 3495 Leu His Pro Met Pro Arg Gly Cys Leu Ala Ser Glu SerSer Glu Gly 1085 1090 1095 cat gta aca ggc tct gag gct gag ctc cag gagaaa gtg tca atg tgt 3543 His Val Thr Gly Ser Glu Ala Glu Leu Gln Glu LysVal Ser Met Cys 1100 1105 1110 1115 aga agc cgg agc agg agc cgg agc ccacgg cca cgc gga gat agc gcc 3591 Arg Ser Arg Ser Arg Ser Arg Ser Pro ArgPro Arg Gly Asp Ser Ala 1120 1125 1130 tac cat tcc cag cgc cac agt ctgctg act cct gtt acc cca ctc tcc 3639 Tyr His Ser Gln Arg His Ser Leu LeuThr Pro Val Thr Pro Leu Ser 1135 1140 1145 cca ccc ggg tta gag gaa gaggat gtc aac ggt tat gtc atg cca gat 3687 Pro Pro Gly Leu Glu Glu Glu AspVal Asn Gly Tyr Val Met Pro Asp 1150 1155 1160 aca cac ctc aaa ggt actccc tcc tcc cgg gaa ggc acc ctt tct tca 3735 Thr His Leu Lys Gly Thr ProSer Ser Arg Glu Gly Thr Leu Ser Ser 1165 1170 1175 gtg ggt ctc agt tctgtc ctg ggt act gaa gaa gaa gat gaa gat gag 3783 Val Gly Leu Ser Ser ValLeu Gly Thr Glu Glu Glu Asp Glu Asp Glu 1180 1185 1190 1195 gag tat gaatac atg aac cgg agg aga agg cac agt cca cct cat ccc 3831 Glu Tyr Glu TyrMet Asn Arg Arg Arg Arg His Ser Pro Pro His Pro 1200 1205 1210 cct aggcca agt tcc ctt gag gag ctg ggt tat gag tac atg gat gtg 3879 Pro Arg ProSer Ser Leu Glu Glu Leu Gly Tyr Glu Tyr Met Asp Val 1215 1220 1225 gggtca gac ctc agt gcc tct ctg ggc agc aca cag agt tgc cca ctc 3927 Gly SerAsp Leu Ser Ala Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu 1230 1235 1240cac cct gta ccc atc atg ccc act gca ggc aca act cca gat gaa gac 3975 HisPro Val Pro Ile Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp 1245 12501255 tat gaa tat atg aat cgg caa cga gat gga ggt ggt cct ggg ggt gat4023 Tyr Glu Tyr Met Asn Arg Gln Arg Asp Gly Gly Gly Pro Gly Gly Asp1260 1265 1270 1275 tat gca gcc atg ggg gcc tgc cca gca tct gag caa gggtat gaa gag 4071 Tyr Ala Ala Met Gly Ala Cys Pro Ala Ser Glu Gln Gly TyrGlu Glu 1280 1285 1290 atg aga gct ttt cag ggg cct gga cat cag gcc ccccat gtc cat tat 4119 Met Arg Ala Phe Gln Gly Pro Gly His Gln Ala Pro HisVal His Tyr 1295 1300 1305 gcc cgc cta aaa act cta cgt agc tta gag gctaca gac tct gcc ttt 4167 Ala Arg Leu Lys Thr Leu Arg Ser Leu Glu Ala ThrAsp Ser Ala Phe 1310 1315 1320 gat aac cct gat tac tgg cat agc agg cttttc ccc aag gct aat gcc 4215 Asp Asn Pro Asp Tyr Trp His Ser Arg Leu PhePro Lys Ala Asn Ala 1325 1330 1335 cag aga acg taa ctcctgctcc ctgtggcactcagggagcat ttaatggcag 4267 Gln Arg Thr 1340 ctagtgcctt tagagggtaccgtcttctcc ctattccctc tctctcccag gtcccagccc 4327 cttttcccca gtcccagacaattccattca atctttggag gcttttaaac attttgacac 4387 aaaattctta tggtatgtagccagctgtgc actttcttct ctttcccaac cccaggaaag 4447 gttttcctta ttttgtgtgctttcccagtc ccattcctca gcttcttcac aggcactcct 4507 ggagatatga aggattactctccatatccc ttcctctcag gctcttgact acttggaact 4567 aggctcttat gtgtgcctttgtttcccatc agactgtcaa gaagaggaaa gggaggaaac 4627 ctagcagagg aaagtgtaattttggtttat gactcttaac cccctagaaa gacagaagct 4687 taaaatctgt gaagaaagaggttaggagta gatattgatt actatcataa ttcagcactt 4747 aactatgagc caggcatcatactaaacttc acctacatta tctcacttag tcctttatca 4807 tccttaaaac aattctgtgacatacatatt atctcatttt acacaaaggg aagtcgggca 4867 tggtggctca tgcctgtaatctcagcactt tgggaggctg aggcagaagg attacctgag 4927 gcaaggagtt tgagaccagcttagccaaca tagtaagacc cccatctc 4975 <210> SEQ ID NO 4 <211> LENGTH: 27<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR Primer <400> SEQUENCE: 4 tcgtcatgtt gaactataacaccaact 27 <210> SEQ ID NO 5 <211> LENGTH: 26 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:PCR Primer <400> SEQUENCE: 5 tgacaaagct tatcgttctt ctcaat 26 <210> SEQID NO 6 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400>SEQUENCE: 6 ccgcttgact cagctcaccg agattc 26 <210> SEQ ID NO 7 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 7caacggattt ggtcgtattg g 21 <210> SEQ ID NO 8 <211> LENGTH: 26 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 8 ggcaacaata tccactttac cagagt26 <210> SEQ ID NO 9 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe<400> SEQUENCE: 9 cgcctggtca ccagggctgc t 21 <210> SEQ ID NO 10 <211>LENGTH: 14796 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (2811)...(2921) <221>NAME/KEY: CDS <222> LOCATION: (3174)...(3283) <221> NAME/KEY: CDS <222>LOCATION: (5158)...(5275) <221> NAME/KEY: CDS <222> LOCATION:(11955)...(12044) <400> SEQUENCE: 10 tctagacatg cggatatatt caagctgggcacagcacagc agccccaccc caggcagctt 60 gaaatcagag ctggggtcca aagggaccacaccccgaggg actgtgtggg ggtcggggca 120 cacaggccac tgcttccccc cgtctttctcagccattcct gaagtcagcc tcactctgct 180 tctcagggat ttcaaatgtg cagagactctggcacttttg tagaagcccc ttctggtcct 240 aacttacacc tggatgctgt ggggctgcagctgctgctcg ggctcgggag gatgctgggg 300 gcccggtgcc catgagcttt tgaagctcctggaactcggt tttgagggtg ttcaggtcca 360 ggtggacacc tgggctgtcc ttgtccatgcatttgatgac attgtgtgca gaagtgaaaa 420 ggagttaggc cgggcatgct ggcttatgcctgtaatccca gcactttggg aggctgaggc 480 gggtggatca cgaggtcagg agttcaataccagcctggcc aagatggtga aaccccgtct 540 ctactaaaaa tacaaaaaaa ttagccgggcatggtggcgg gcgcatgtaa tcccagctac 600 tgggggggct gaggcagaga attgctggaacccaggagat ggaggttgca gtgagccaag 660 attgtgccac tgcactgcac tccagcctggcgacagagca agactctgtc tcaaaaaaaa 720 aaaaaaaaag tgaaaaggag ttgttcctttcctccctcct gagggcaggc aactgctgcg 780 gttgccagtg gaggtggtgc gtccttggtctgtgcctggg ggccacccca gcagaggcca 840 tggtggtgcc agggcccggt tagcgagccaatcagcagga cccaggggcg acctgccaaa 900 gtcaactgga tttgataact gcagcgaagttaagtttcct gattttgatg attgtgttgt 960 ggttgtgtaa gagaatgaag tatttcggggtagtatggta atgccttcaa cttacaaacg 1020 gttcaggtaa accacccata tacatacatatacatgcatg tgatatatac acatacaggg 1080 atgtgtgtgt gttcacatat atgaggggagagagactagg ggagagaaag taggttgggg 1140 agagggagag agaaaggaaa acaggagacagagagagagc ggggagtaga gagagggaag 1200 gggtaagaga gggagaggag gagagaaagggaggaagaag cagagagtga atgttaaagg 1260 aaacaggcaa aacataaaca gaaaatctgggtgaagggta tatgagtatt ctttgtacta 1320 ttcttgcaat tatcttttat ttaaattgacatcgggccgg gcgcagtggc tcacatctgt 1380 aatcccagca ctttgggagg ccgaggcaggcagatcactt gaggtcagga gtttgagacc 1440 agcctggcaa acatggtgaa accccatctctactaaaaat acaaaaatta gcctggtgtg 1500 gtggtgcatg cctttaatct cagctactcgggaggctgag gcaggagaat cgcttgaacc 1560 cgtggcgggg aggaggttgc agtgagctgagatcatgcca ctgcactcca gcctgggcga 1620 tagagcgaga ctcagtttca aataaataaataaacatcaa aataaaaagt tactgtatta 1680 aagaatgggg gcggggtggg aggggtggggagaggttgca aaaataaata aataaataaa 1740 taaaccccaa aatgaaaaag acagtggaggcaccaggcct gcgtggggct ggagggctaa 1800 taaggccagg cctcttatct ctggccatagaaccagagaa gtgagtggat gtgatgccca 1860 gctccagaag tgactccaga acaccctgttccaaagcaga ggacacactg attttttttt 1920 taataggctg caggacttac tgttggtgggacgccctgct ttgcgaaggg aaaggaggag 1980 tttgccctga gcacaggccc ccaccctccactgggctttc cccagctccc ttgtcttctt 2040 atcacggtag tggcccagtc cctggcccctgactccagaa ggtggccctc ctggaaaccc 2100 aggtcgtgca gtcaacgatg tactcgccgggacagcgatg tctgctgcac tccatccctc 2160 ccctgttcat ttgtccttca tgcccgtctggagtagatgc tttttgcaga ggtggcaccc 2220 tgtaaagctc tcctgtctga ctttttttttttttttagac tgagttttgc tcttgttgcc 2280 taggctggag tgcaatggca caatctcagctcactgcacc ctctgcctcc cgggttcaag 2340 cgattctcct gcctcagcct cccgagtagttgggattaca ggcatgcacc accacgccca 2400 gctaattttt gtatttttag tagagacaaggtttcaccgt gatggccagg ctggtcttga 2460 actccaggac tcaagtgatg ctcctgcctaggcctctcaa agtgttggga ttacaggcgt 2520 gagccactgc acccggcctg cacgcgttctttgaaagcag tcgagggggc gctaggtgtg 2580 ggcagggacg agctggcgcg gcgtcgctgggtgcaccgcg accacgggca gagccacgcg 2640 gcgggaggac tacaactccc ggcacaccccgcgccgcccc gcctctactc ccagaaggcc 2700 gcggggggtg gaccgcctaa gagggcgtgcgctcccgaca tgccccgcgg cgcgccatta 2760 accgccagat ttgaatcgcg ggacccgttggcagaggtgg cggcggcggc atg ggt 2816 Met Gly 1 gcc ccg acg ttg ccc cct gcctgg cag ccc ttt ctc aag gac cac cgc 2864 Ala Pro Thr Leu Pro Pro Ala TrpGln Pro Phe Leu Lys Asp His Arg 5 10 15 atc tct aca ttc aag aac tgg cccttc ttg gag ggc tgc gcc tgc acc 2912 Ile Ser Thr Phe Lys Asn Trp Pro PheLeu Glu Gly Cys Ala Cys Thr 20 25 30 ccg gag cgg gtgagactgc ccggcctcctggggtccccc acgcccgcct tgccctgtcc 2971 Pro Glu Arg 35 ctagcgaggccactgtgact gggcctcggg ggtacaagcc gccctcccct ccccgtcctg 3031 tccccagcgaggccactgtg gctgggcccc ttgggtccag gccggcctcc cctccctgct 3091 ttgtccccatcgaggccttt gtggctgggc ctcggggttc cgggctgcca cgtccactca 3151 cgagctgtgctgtcccttgc ag atg gcc gag gct ggc ttc atc cac tgc ccc 3203 Met Ala GluAla Gly Phe Ile His Cys Pro 40 45 act gag aac gag cca gac ttg gcc cagtgt ttc ttc tgc ttc aag gag 3251 Thr Glu Asn Glu Pro Asp Leu Ala Gln CysPhe Phe Cys Phe Lys Glu 50 55 60 ctg gaa ggc tgg gag cca gat gac gac cccat gtaagtcttc tctggccagc 3303 Leu Glu Gly Trp Glu Pro Asp Asp Asp ProIle 65 70 ctcgatgggc tttgttttga actgagttgt caaaagattt gagttgcaaagacacttagt 3363 atgggagggt tgctttccac cctcattgct tcttaaacag ctgttgtgaacggatacctc 3423 tctatatgct ggtgccttgg tgatgcttac aacctaatta aatctcatttgaccaaaatg 3483 ccttggggtg gacgtaagat gcctgatgcc tttcatgttc aacagaatacatcagcagac 3543 cctgttgttg tgaactccca ggaatgtcca agtgcttttt ttgagattttttaaaaaaca 3603 gtttaattga aatataacct acacagcaca aaaattaccc tttgaaagtgtgcacttcac 3663 actttcggag gctgaggcgg gcggatcacc tgaggtcagg agttcaagacctgcctggcc 3723 aacttggcga aaccccgtct ctactaaaaa tacaaaaatt agccgggcatggtagcgcac 3783 gcccgtaatc ccagctactc gggaggctaa ggcaggagaa tcgcttgaacctgggaggcg 3843 gaggttgcag tgagccgaga ttgtgccaat gcactccagc ctcggcgacagagcgagact 3903 ccgtcataaa aataaaaaat tgaaaaaaaa aaaagaaaga aagcatatacttcagtgttg 3963 ttctggattt ttttcttcaa gatgcctagt taatgacaat gaaattctgtactcggatgg 4023 tatctgtctt tccacactgt aatgccatat tcttttctca cctttttttctgtcggattc 4083 agttgcttcc acagctttaa tttttttccc ctggagaatc accccagttgtttttctttt 4143 tggccagaag agagtagctg ttttttttct tagtatgttt gctatggtggttatactgca 4203 tccccgtaat cactgggaaa agatcagtgg tattcttctt gaaaatgaataagtgttatg 4263 atattttcag attagagtta caactggctg tctttttgga ctttgtgtggccatgttttc 4323 attgtaatgc agttctggta acggtgatag tcagttatac agggagactcccctagcaga 4383 aaatgagagt gtgagctagg gggtcccttg gggaacccgg ggcaataatgcccttctctg 4443 cccttaatcc ttacagtggg ccgggcacgg tggcttacgc ctgtaataccagcactttgg 4503 gaggccgagg cgggcggatc acgaggtcag gagatcgaga ccatcttggctaatacggtg 4563 aaaccccgtc tccactaaaa atacaaaaaa ttagccgggc gtggtggtgggcgcctgtag 4623 tcccagctac tcgggaggct gaggcaggag aatggcgtga acccaggaggcggagcttgc 4683 agtgagccga gattgcacca ctgcactcca gcctgggcga cagaatgagactccgtctca 4743 aaaaaaaaaa aaaaagaaaa aaatctttac agtggattac ataacaattccagtgaaatg 4803 aaattacttc aaacagttcc ttgagaatgt tggagggatt tgacatgtaattcctttgga 4863 catataccat gtaacacttt tccaactaat tgctaaggaa gtccagataaaatagataca 4923 ttagccacac agatgtgggg ggagatgtcc acagggagag agaaggtgctaagaggtgcc 4983 atatgggaat gtggcttggg caaagcactg atgccatcaa cttcagacttgacgtcttac 5043 tcctgaggca gagcagggtg tgcctgtgga gggcgtgggg aggtggcccgtggggagtgg 5103 actgccgctt taatcccttc agctgccttt ccgctgttgt tttgatttttctag a gag 5161 Glu 75 gaa cat aaa aag cat tcg tcc ggt tgc gct ttc ctttct gtc aag aag 5209 Glu His Lys Lys His Ser Ser Gly Cys Ala Phe Leu SerVal Lys Lys 80 85 90 cag ttt gaa gaa tta acc ctt ggt gaa ttt ttg aaa ctggac aga gaa 5257 Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu AspArg Glu 95 100 105 aga gcc aag aac aaa att gtatgtattg ggaataagaactgctcaaac cctgttcaat 5315 Arg Ala Lys Asn Lys Ile 110 gtctttagcactaaactacc tagtccctca aagggactct gtgttttcct caggaagcat 5375 tttttttttttttctgagat agagtttcac tcttgttgcc caggctggag tgcaatggtg 5435 caatcttggctcactgcaac ctctgcctct cgggttcaag tgattctcct gcctcagcct 5495 cccaagtaactgggattaca gggaagtgcc accacaccca gctaattttt gtatttttag 5555 tagagatggggtttcaccac attgcccagg ctggtcttga actcctgacc tcgtgattcg 5615 cccaccttggcctcccaaag tgctgggatt acaggcgtga accaccacgc ctggcttttt 5675 tttttttgttctgagacaca gtttcactct gttacccagg ctggagtagg gtggcctgat 5735 ctcggatcactgcaacctcc gcctcctggg ctcaagtgat ttgcctgctt cagcctccca 5795 agtagccgagattacaggca tgtgccacca cacccaggta atttttgtat ttttggtaga 5855 gacgaggtttcaccatgttg gccaggctgg ttttgaactc ctgacctcag gtgatccacc 5915 cgcctcagcctcccaaagtg ctgagattat aggtgtgagc caccacacct ggcctcagga 5975 agtatttttatttttaaatt tatttattta tttgagatgg agtcttgctc tgtcgcccag 6035 gctagagtgcagcgacggga tctcggctca ctgcaagctc cgccccccag gttcaagcca 6095 ttctcctgcctcagcctccc gagtagctgg gactacaggc gcccgccacc acacccggct 6155 aatttttttgtatttttagt agagacgggt tttcaccgtg ttagccagga gggtcttgat 6215 ctcctgacctcgtgatctgc ctgcctcggc ctcccaaagt gctgggatta caggtgtgag 6275 ccaccacacccggctatttt tatttttttg agacagggac tcactctgtc acctgggctg 6335 cagtgcagtggtacaccata gctcactgca gcctcgaact cctgagctca agtgatcctc 6395 ccacctcatcctcacaagta attgggacta caggtgcacc ccaccatgcc cacctaattt 6455 atttatttatttatttattt attttcatag agatgagggt tccctgtgtt gtccaggctg 6515 gtcttgaactcctgagctca cgggatcctt ttgcctgggc ctcccaaagt gctgagatta 6575 caggcatgagccaccgtgcc cagctaggaa tcatttttaa agcccctagg atgtctgtgt 6635 gattttaaagctcctggagt gtggccggta taagtatata ccggtataag taaatcccac 6695 attttgtgtcagtatttact agaaacttag tcatttatct gaagttgaaa tgtaactggg 6755 ctttatttatttatttattt atttatttat ttttaatttt tttttttgag acgagtctca 6815 ctttgtcacccaggctggag tgcagtggca cgatctcggc tcactgcaac ctctgcctcc 6875 cggggtcaagcgattctcct gccttagcct cccgagtagc tgggactaca ggcacgcacc 6935 accatgcctggctaattttt gtatttttag tagacggggt ttcaccatgc tggccaagct 6995 ggtctcaaactcctgacctt gtgatctgcc cgctttagcc tcccagagtg ctgggattac 7055 aggcatgagccaccatgcgt ggtcttttta aaattttttg attttttttt tttttgagac 7115 agagccttgctctgtcgccc aggctggagt gcagtggcac gatctcagct cactacaagc 7175 tccgcctcccgggttcacgc cattcttctg cctcagcctc ctgagtagct gggactacag 7235 gtgcccaccaccacgcctgg ctaatttttt ttggtatttt tattagagac aaggtttcat 7295 catgttggccaggctggtct caaactcctg acctcaagtg atctgcctgc ctcggcctcc 7355 caaagcgctgagattacagg tgtgatctac tgcgccaggc ctgggcgtca tatattctta 7415 tttgctaagtctggcagccc cacacagaat aagtactggg ggattccata tccttgtagc 7475 aaagccctgggtggagagtc aggagatgtt gtagttctgt ctctgccact tgcagacttt 7535 gagtttaagccagtcgtgct catgctttcc ttgctaaata gaggttagac cccctatccc 7595 atggtttctcaggttgcttt tcagcttgaa aattgtattc ctttgtagag atcagcgtaa 7655 aataattctgtccttatatg tggctttatt ttaatttgag acagagtgtc actcagtcgc 7715 ccaggctggagtgtggtggt gcgatcttgg ctcactgcga cctccacctc ccaggttcaa 7775 gcgattctcgtgcctcaggc tcccaagtag ctgagattat aggtgtgtgc caccaggccc 7835 agctaacttttgtattttta gtagagacag ggttttgcca tgttggctaa gctggtctcg 7895 aactcctggcctcaagtgat ctgcccgcct tggcatccca aagtgctggg attacaggtg 7955 tgaaccaccacacctggcct caatatagtg gcttttaagt gctaaggact gagattgtgt 8015 tttgtcaggaagaggccagt tgtgggtgaa gcatgctgtg agagagcttg tcacctggtt 8075 gaggttgtgggagctgcagc gtgggaactg gaaagtgggc tggggatcat ctttttccag 8135 gtcaggggtcagccagcttt tctgcagcgt gccatagacc atctcttagc cctcgtgggt 8195 cagagtctctgttgcatatt gtcttttgtt gtttttcaca accttttaga aacataaaaa 8255 gcattcttagcccgtgggct ggacaaaaaa aggccatgac gggctgtatg gatttggccc 8315 agcaggcccttgcttgccaa gccctgtttt agacaaggag cagcttgtgt gcctggaacc 8375 atcatgggcacaggggagga gcagagtgga tgtggaggtg tgagctggaa accaggtccc 8435 agagcgctgagaaagacaga gggtttttgc ccttgcaagt agagcaactg aaatctgaca 8495 ccatccagttccagaaagcc ctgaagtgct ggtggacgct gcggggtgct ccgctctagg 8555 gttacagggatgaagatgca gtctggtagg gggagtccac tcacctgttg gaagatgtga 8615 ttaagaaaagtagactttca gggccgggca tggtggctca cgcctgtaat cccagcactt 8675 tgggaggccgaggcgggtgg atcacgaggt caggagatcg agaccatcct ggctaacatg 8735 gtgaaaccccgtctttacta aaaatacaaa aaattagctg ggcgtggtgg cgggcgcctg 8795 tagtcccagctactcgggag gctgaggcag gagaatggcg tgaacctggg aggtggagct 8855 tgctgtgagccgagatcgcg ccactgcact ccagcctggg cgacagagcg agactccgtc 8915 tcaaaaaaaaaaaaaaaagt aggctttcat gatgtgtgag ctgaaggcgc agtaggcaga 8975 agtagaggcctcagtccctg caggagaccc ctcggtctct atctcctgat agtcagaccc 9035 agccacactggaaagagggg agacattaca gcctgcgaga aaagtaggga gatttaaaaa 9095 ctgcttggcttttattttga actgtttttt ttgtttgttt gttttcccca attcagaata 9155 cagaatacttttatggattt gtttttatta ctttaatttt gaaacaatat aatctttttt 9215 ttgttgtttttttgagacag ggtcttactc tgtcacccag gctgagtgca gtggtgtgat 9275 cttggctcacctcagcctcg accccctggg ctcaaatgat tctcccacct cagcttccca 9335 agtagctgggaccacaggtg cgtgtgttgc gctatacaaa tcctgaagac aaggatgctg 9395 ttgctggtgatgctggggat tcccaagatc ccagatttga tggcaggatg cccctgtctg 9455 ctgccttgccagggtgccag gagggcgctg ctgtggaagc tgaggcccgg ccatccaggg 9515 cgatgcattgggcgctgatt cttgttcctg ctgctgcctc ggtgcttagc ttttgaaaca 9575 atgaaataaattagaaccag tgtgaaaatc gatcagggaa taaatttaat gtggaaataa 9635 actgaacaacttagttcttc ataagagttt acttggtaaa tacttgtgat gaggacaaaa 9695 cgaagcactagaaggagagg cgagttgtag acctgggtgg caggagtgtt ttgtttgttt 9755 tctttggcagggtcttgctc tgttgctcag gctggagtac agtggcacaa tcacagctca 9815 ctatagcctcgacctcctgg actcaagcaa tcctcctgcc tcagcctccc agtagctggg 9875 actacaggcgcatgccacca tgcctggcta attttaaatt tttttttttc tcttttttga 9935 gatggaatctcactctgtcg cccaggctgg agtgcagtgg cgtgatctcg gctgacggca 9995 agctccgcctcccaggttca ctccattcgc ctgcctcagc ctcccaagta gctgggacta 10055 caggcgctgggattacaaac ccaaacccaa agtgctggga ttacaggcgt gagccactgc 10115 acccggcctgttttgtcttt caatagcaag agttgtgttt gcttcgcccc tacctttagt 10175 ggaaaaatgtataaaatgga gatattgacc tccacattgg ggtggttaaa ttatagcatg 10235 tatgcaaaggagcttcgcta atttaaggct tttttgaaag agaagaaact gaataatcca 10295 tgtgtgtatatatattttaa aagccatggt catctttcca tatcagtaaa gctgaggctc 10355 cctgggactgcagagttgtc catcacagtc cattataagt gcgctgctgg gccaggtgca 10415 gtggcttgtgcctgaatccc agcactttgg gaggccaagg caggaggatt cattgagccc 10475 aggagttttgaggcgagcct gggcaatgtg gccagacctc atctcttcaa aaaatacaca 10535 aaaaattagccaggcatggt ggcacgtgcc tgtagtctca gctactcagg aggctgaggt 10595 gggaggatcactttgagcct tgcaggtcaa agctgcagta agccatgatc ttgccactgc 10655 attccagcctggatgacaga gcgagaccct gtctctaaaa aaaaaaaaaa ccaaacggtg 10715 cactgttttcttttttctta tcaatttatt atttttaaat taaattttct tttaataatt 10775 tataaattataaatttatat taaaaaatga caaattttta ttacttatac atgaggtaaa 10835 acttaggatatataaagtac atattgaaaa gtaatttttt ggctggcaca gtggctcaca 10895 cctgtaatcccagcactttg ggaggccgtg gcgggcagat cacatgagat catgagttcg 10955 agaccaacctgaccaacatg gagagacccc atctctacta aaaatacaaa attagccggg 11015 gtggtggcgcatgcctgtaa tcccagctac tcgggaggct gaggcaggag aatctcttga 11075 acccgggaggcagaggttgc ggtgagccaa gatcgtgcct ttgcacacca gcctaggcaa 11135 caagagcgaaagtccgtctc aaaaaaaaag taattttttt taagttaacc tctgtcagca 11195 aacaaatttaacccaataaa ggtctttgtt ttttaatgta gtagaggagt tagggtttat 11255 aaaaaatatggtagggaagg gggtccctgg atttgctaat gtgattgtca tttgcccctt 11315 aggagagagctctgttagca gaatgaaaaa attggaagcc agattcaggg agggactgga 11375 agcaaaagaatttctgttcg aggaagagcc tgatgtttgc cagggtctgt ttaactggac 11435 atgaagaggaaggctctgga ctttcctcca ggagtttcag gagaaaggta gggcagtggt 11495 taagagcagagctctgccta gactagctgg ggtgcctaga ctagctgggg tgcccagact 11555 agctggggtgcctagactag ctgggtactt tgagtggctc cttcagcctg gacctcggtt 11615 tcctcacctgtatagtagag atatgggagc acccagcgca ggatcactgt gaacataaat 11675 cagttaatggaggaagcagg tagagtggtg ctgggtgcat accaagcact ccgtcagtgt 11735 ttcctgttattcgatgatta ggaggcagct taaactagag ggagttgagc tgaatcagga 11795 tgtttgtcccaggtagctgg gaatctgcct agcccagtgc ccagtttatt taggtgctct 11855 ctcagtgttccctgattgtt ttttcctttg tcatcttatc tacaggatgt gactgggaag 11915 ctctggtttcagtgtcatgt gtctattctt tatttccag gca aag gaa acc aac 11969 Ala Lys GluThr Asn 115 aat aag aag aaa gaa ttt gag gaa act gcg aag aaa gtg cgc cgtgcc 12017 Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala Lys Lys Val Arg ArgAla 120 125 130 atc gag cag ctg gct gcc atg gat tga ggcctctggccggagctgcc 12064 Ile Glu Gln Leu Ala Ala Met Asp 135 140 tggtcccagagtggctgcac cacttccagg gtttattccc tggtgccacc agccttcctg 12124 tgggccccttagcaatgtct taggaaagga gatcaacatt ttcaaattag atgtttcaac 12184 tgtgctcctgttttgtcttg aaagtggcac cagaggtgct tctgcctgtg cagcgggtgc 12244 tgctggtaacagtggctgct tctctctctc tctctctttt ttgggggctc atttttgctg 12304 ttttgattcccgggcttacc aggtgagaag tgagggagga agaaggcagt gtcccttttg 12364 ctagagctgacagctttgtt cgcgtgggca gagccttcca cagtgaatgt gtctggacct 12424 catgttgttgaggctgtcac agtcctgagt gtggacttgg caggtgcctg ttgaatctga 12484 gctgcaggttccttatctgt cacacctgtg cctcctcaga ggacagtttt tttgttgttg 12544 tgtttttttgtttttttttt ttggtagatg catgacttgt gtgtgatgag agaatggaga 12604 cagagtccctggctcctcta ctgtttaaca acatggcttt cttattttgt ttgaattgtt 12664 aattcacagaatagcacaaa ctacaattaa aactaagcac aaagccattc taagtcattg 12724 gggaaacggggtgaacttca ggtggatgag gagacagaat agagtgatag gaagcgtctg 12784 gcagatactccttttgccac tgctgtgtga ttagacaggc ccagtgagcc gcggggcaca 12844 tgctggccgctcctccctca gaaaaaggca gtggcctaaa tcctttttaa atgacttggc 12904 tcgatgctgtgggggactgg ctgggctgct gcaggccgtg tgtctgtcag cccaaccttc 12964 acatctgtcacgttctccac acgggggaga gacgcagtcc gcccaggtcc ccgctttctt 13024 tggaggcagcagctcccgca gggctgaagt ctggcgtaag atgatggatt tgattcgccc 13084 tcctccctgtcatagagctg cagggtggat tgttacagct tcgctggaaa cctctggagg 13144 tcatctcggctgttcctgag aaataaaaag cctgtcattt caaacactgc tgtggaccct 13204 actgggtttttaaaatattg tcagtttttc atcgtcgtcc ctagcctgcc aacagccatc 13264 tgcccagacagccgcagtga ggatgagcgt cctggcagag acgcagttgt ctctgggcgc 13324 ttgccagagccacgaacccc agacctgttt gtatcatccg ggctccttcc gggcagaaac 13384 aactgaaaatgcacttcaga cccacttatt tatgccacat ctgagtcggc ctgagataga 13444 cttttccctctaaactggga gaatatcaca gtggtttttg ttagcagaaa atgcactcca 13504 gcctctgtactcatctaagc tgcttatttt tgatatttgt gtcagtctgt aaatggatac 13564 ttcactttaataactgttgc ttagtaattg gctttgtaga gaagctggaa aaaaatggtt 13624 ttgtcttcaactcctttgca tgccaggcgg tgatgtggat ctcggcttct gtgagcctgt 13684 gctgtgggcagggctgagct ggagccgccc ctctcagccc gcctgccacg gcctttcctt 13744 aaaggccatccttaaaacca gaccctcatg gctgccagca cctgaaagct tcctcgacat 13804 ctgttaataaagccgtaggc ccttgtctaa gcgcaaccgc ctagactttc tttcagatac 13864 atgtccacatgtccattttt caggttctct aagttggagt ggagtctggg aagggttgtg 13924 aatgaggcttctgggctatg ggtgaggttc caatggcagg ttagagcccc tcgggccaac 13984 tgccatcctggaaagtagag acagcagtgc ccgctgccca gaagagacca gcaagccaaa 14044 ctggagcccccattgcaggc tgtcgccatg tggaaagagt aactcacaat tgccaataaa 14104 gtctcatgtggttttatcta cttttttttt ctttttcttt ttttttgaga caaggccttg 14164 ccctcccaggctggagtgca gtggaatgac cacagctcac cgcaacctca aattcttgcg 14224 ttcaagtgaacctcccactt tagcctccca agtagctggg actacaggcg cacgccatca 14284 cacccggctaattgaaaaat tttttttttt gtttagatgg aatctcactt tgttgcccag 14344 gctggtctcaaactcctggg ctcaagtgat catcctgctt cagcgtccga cttgttggta 14404 ttataggcgtgagccactgg gcctgaccta gctaccattt tttaatgcag aaatgaagac 14464 ttgtagaaatgaaataactt gtccaggata gtcgaataag taacttttag agctgggatt 14524 tgaacccaggcaatctggct ccagagctgg gccctcactg ctgaaggaca ctgtcagctt 14584 gggagggtggctatggtcgg ctgtctgatt ctagggagtg agggctgtct ttaaagcacc 14644 ccattccattttcagacagc tttgtcagaa aggctgtcat atggagctga cacctgcctc 14704 cccaaggcttccatagatcc tctctgtaca ttgtaacctt ttattttgaa atgaaaattc 14764 acaggaagttgtaaggctag tacaggggat cc 14796 <210> SEQ ID NO 11 <211> LENGTH: 18 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 11 cgacggcagcggaggttg 18 <210> SEQ ID NO 12 <211> LENGTH: 18 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Antisense Oligonucleotide <400> SEQUENCE: 12 aagagccacc tgaaccgc 18<210> SEQ ID NO 13 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 13 cgcagagggt gaagggag 18 <210> SEQ IDNO 14 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 14 gttgcccacc tcggagcc 18 <210> SEQ IDNO 15 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 15 acactgcctg agagttgc 18 <210> SEQ IDNO 16 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 16 gtgtctggta ttggttct 18 <210> SEQ IDNO 17 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 17 atcaccacct cacacctc 18 <210> SEQ IDNO 18 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 18 tgtcccgtga gcacaatc 18 <210> SEQ IDNO 19 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 19 gaggtcggca ttgtgtcc 18 <210> SEQ IDNO 20 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 20 tgcaggaagg agaggtcg 18 <210> SEQ IDNO 21 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 21 gcggaggttg ggcaatgg 18 <210> SEQ IDNO 22 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 22 cgtggctgga gttggtgt 18 <210> SEQ IDNO 23 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 23 cctgacagaa tctcggtg 18 <210> SEQ IDNO 24 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 24 ggtccctcac gatgtccc 18 <210> SEQ IDNO 25 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 25 cactatctca gcatctcg 18 <210> SEQ IDNO 26 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 26 ccattgtcct tcaccact 18 <210> SEQ IDNO 27 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 27 aggaccccag catcgccc 18 <210> SEQ IDNO 28 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 28 tcaatgtctg gcagtctt 18 <210> SEQ IDNO 29 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 29 gcacagatgg tcttggtc 18 <210> SEQ IDNO 30 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 30 ccattacact gaggagca 18 <210> SEQ IDNO 31 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 31 catggcagca ctggttgg 18 <210> SEQ IDNO 32 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 32 caggcaaagc agtctgtg 18 <210> SEQ IDNO 33 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 33 tgtagacaag aggctgtg 18 <210> SEQ IDNO 34 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 34 acaaactcct ccatactg 18 <210> SEQ IDNO 35 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 35 ctgacacagg atgtttga 18 <210> SEQ IDNO 36 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 36 acatcttgag cccatttt 18 <210> SEQ IDNO 37 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 37 ccagagcctg ttccctca 18 <210> SEQ IDNO 38 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 38 ttcacaaatc catcaatg 18 <210> SEQ IDNO 39 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 39 gactggatgt tcaggtaa 18 <210> SEQ IDNO 40 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 40 cggttgtaga ggcttctg 18 <210> SEQ IDNO 41 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 41 tcggaagccc agagatgt 18 <210> SEQ IDNO 42 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 42 agagtggtgg tagcagag 18 <210> SEQ IDNO 43 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 43 aagcaccttg gtccagtt 18 <210> SEQ IDNO 44 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 44 ggacaagcac tgaccagg 18 <210> SEQ IDNO 45 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 45 tggcaggaga agcattcg 18 <210> SEQ IDNO 46 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 46 acaagtatca gagcccga 18 <210> SEQ IDNO 47 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 47 gggcacattg agcacaag 18 <210> SEQ IDNO 48 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 48 tgtcctaaac agtcttga 18 <210> SEQ IDNO 49 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 49 tgtcagatgg gttttgcc 18 <210> SEQ IDNO 50 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 50 cctgctatca ctgtcaaa 18 <210> SEQ IDNO 51 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 51 aatcactacc aatcctgc 18 <210> SEQ IDNO 52 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 52 ccagtagaga aaagtgcc 18 <210> SEQ IDNO 53 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 53 cctcatagcc cttttatt 18 <210> SEQ IDNO 54 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 54 ccccgttcca agtatcgc 18 <210> SEQ IDNO 55 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 55 tatgctctca ccccgttc 18 <210> SEQ IDNO 56 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 56 gactttgtta gccttctc 18 <210> SEQ IDNO 57 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 57 agacacccga gccaagca 18 <210> SEQ IDNO 58 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 58 ttgattgatt caccctca 18 <210> SEQ IDNO 59 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 59 ccactcttgt cctcaatg 18 <210> SEQ IDNO 60 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 60 aaaactctgc cgtccact 18 <210> SEQ IDNO 61 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 61 tagtcccagc agccttac 18 <210> SEQ IDNO 62 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 62 gagaacccag aggcaaat 18 <210> SEQ IDNO 63 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 63 ctccccagtt gagcagca 18 <210> SEQ IDNO 64 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 64 cgggcagcca ggtttcta 18 <210> SEQ IDNO 65 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 65 cacctgaacc tgactggg 18 <210> SEQ IDNO 66 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 66 caccaaaatc tgccacct 18 <210> SEQ IDNO 67 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 67 atcatcagga ggcagcag 18 <210> SEQ IDNO 68 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 68 cccaaagtgg atactctc 18 <210> SEQ IDNO 69 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 69 caactcccaa actgtcac 18 <210> SEQ IDNO 70 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 70 ccccgaaggt catcaact 18 <210> SEQ IDNO 71 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 71 ggtacttcag ccaatcgt 18 <210> SEQ IDNO 72 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 72 atcaatcatc caacactt 18 <210> SEQ IDNO 73 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 73 gggcgaatgt tctcatca 18 <210> SEQ IDNO 74 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 74 cttcttgttt gtcagacc 18 <210> SEQ IDNO 75 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 75 gtgttccaac tggtaggc 18 <210> SEQ IDNO 76 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 76 gtggcattgg gtgtagag 18 <210> SEQ IDNO 77 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 77 acattgacac tttctcct 18 <210> SEQ IDNO 78 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 78 ccgttgacat cctcttcc 18 <210> SEQ IDNO 79 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 79 gacagaactg agacccac 18 <210> SEQ IDNO 80 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 80 attcatactc ctcatctt 18 <210> SEQ IDNO 81 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 81 tcttcatctg gagttgtg 18 <210> SEQ IDNO 82 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 82 ttcataccct tgctcaga 18 <210> SEQ IDNO 83 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 83 tcagggttat caaaggca 18 <210> SEQ IDNO 84 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 84 tacgttctct gggcatta 18 <210> SEQ IDNO 85 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 85 agcctccaaa gattgaat 18 <210> SEQ IDNO 86 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 86 ggaatgggac tgggaaag 18 <210> SEQ IDNO 87 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 87 acaaaggcac acataaga 18 <210> SEQ IDNO 88 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 88 ccctttcctc ttcttgac 18 <210> SEQ IDNO 89 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 89 tcctaacctc tttcttca 18 <210> SEQ IDNO 90 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 90 gtaggtgaag tttagtat 18 <210> SEQ IDNO 91 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 91 aggatgataa aggactaa 18 <210> SEQ IDNO 92 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 92 tggtctcaaa ctccttgc 18 <210> SEQ IDNO 93 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 93 gggtcttact atgttggc 18 <210> SEQ IDNO 94 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 94 caaagtgctg agattaca 18

What is claimed is:
 1. An antisense compound up to 50 nucleobases inlength comprising at least an 8-nucleobase portion of SEQ ID NOS: 11,12, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 39, 41, 43, 44, 45, 46, 48, 49, 52, 53, 54, 56, 57,58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 76, 77, 78,79, 81, 82, 83, 84, 85, 87, 88, 90, 92, 93 or 94 which inhibits theexpression of human Her-3.
 2. An antisense compound up to 50 nucleobasesin length comprising SEQ ID NOS: 18 or 63 which inhibits the expressionof human Her-3.
 3. The antisense compound of claim 1 or claim 2 which isan antisense oligonucleotide.
 4. The antisense compound of claim 3wherein the antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 5. The antisense compound of claim 4 whereinthe modified internucleoside linkage is a phosphorothioate linkage. 6.The antisense compound of claim 3 wherein the antisense oligonucleotidecomprises at least one modified sugar moiety.
 7. The antisense compoundof claim 6 wherein the modified sugar moiety is a 2′-O-methoxyethylsugar moiety.
 8. The antisense compound of claim 3 wherein the antisenseoligonucleotide comprises at least one modified nucleobase.
 9. Theantisense compound of claim 8 wherein the modified nucleobase is a5-methylcytosine.
 10. The antisense compound of claim 3 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 11. A method ofinhibiting the expression of human Her-3 in human cells or tissuescomprising contacting said cells or tissues in vitro with the antisensecompound of claim 1 or 2 so that expression of human Here-3 isinhibited.